Dietary overlap and selectivity among mountain steppe river fish in the United States and Mongolia

Abstract Lotic systems in mountain regions have historically provided secure habitat for native fish populations because of their relative isolation from human settlement and lack of upstream disturbances. However, rivers of mountain ecoregions are currently experiencing heightened levels of disturbance due to the introduction of nonnative species impacting endemic fishes in these areas. We compared the fish assemblages and diets of mountain steppe fishes of the stocked rivers in Wyoming with rivers in northern Mongolia where stocking is absent. Using gut content analysis, we quantified the selectivity and diets of fishes collected in these systems. Nonnative species had more generalist diets with lower levels of selectivity than most native species and native species had high levels of dietary specificity and selectivity. High abundances of nonnative species and high levels of dietary overlaps in our Wyoming sites is a cause of concern for native Cutthroat Trout and overall system stability. In contrast, fish assemblages characterizing Mongolia mountain steppe rivers were composed of only native species with diverse diets and higher selectivity values, suggesting low probability for interspecific competition.


| INTRODUC TI ON
Lotic systems in mountain regions have historically provided secure habitat for native fish populations because of their relative isolation from human settlement and lack of upstream disturbances (Adams et al., 2001;Isaak et al., 2016). However, rivers of mountain ecoregions are currently experiencing heightened levels of anthropogenic disturbance (Hofmann et al., 2015;Leu et al., 2008;Wohl, 2006).
In the expansive mountain steppe ecoregions characterizing the mountain regions of western United States (US) and northern Mongolia, multiple anthropogenic pressures have the potential to impact endemic fish species (Kaus et al., 2019). In the mountain steppe rivers of Mongolia, fishes started to be exposed to nonnative species (Mendsaikhan et al., 2017) and impacts caused by mining and free-range livestock grazing (Chalov et al., 2012). In western US mountain steppe rivers, fishes are impacted by beaver removal, habitat alterations from mining activities, and the introduction of nonnative fishes (McKelvey et al., 2016;Wohl, 2006). Stocking hatchery-raised native and nonnative salmonid species can be detrimental to wild fishes and associated assemblages due to increased competition for food, potential for hybridization and predation of native fishes (Seiler & Keeley, 2009).
Gut content analysis is a low cost and informative method of dietary analysis which provides details on a range of dietary information (Baker et al., 2014;Declerck et al., 2002;Pilger et al., 2010).
By determining the degree of dietary overlap among species, we can predict potential impacts of introduced species on native species (Declerck et al., 2002;Pilger et al., 2010;Sampson et al., 2009).
Identification of specific dietary items, though the use of gut content analysis, also allows for the calculation of selectivity indices identifying diet item preferences, key dietary components and how these preferences may change based on biotic (seasonal fluctuations in diet populations, interactions with other fish species, etc.) and abiotic (elevation, temperature, flow, etc.) factors (Hilderbrand & Kershner, 2004;Lowe et al., 2000;Mischke et al., 2003;Nakano et al., 1999).
The research presented here is a small part of a larger macrosystem ecology project comparing rivers in the Western United States and Mongolia which contrasted in their level of human impact. The aim of this project was to compare the fish assemblages and diets of mountain steppe fishes of the stocked, heavily managed rivers of the western United States and rivers in northern Mongolia where stocking is absent. Understanding how diets differ in different fish assemblages based on the presence of stocked nonnative fishes allows us to predict potential impacts on recently altered systems.
We hypothesized that (1) in Wyoming, where native and nonnative species overlap, nonnative species will have generalist diets without high levels of selectivity and native species will have specialized diets with higher levels of selectivity; and (2) in Mongolia where nonnative fishes are not well established, native fishes will have specialized diets and high dietary selectivity, and (3) Wyoming fish assemblages will have higher dietary overlaps compared to those of Mongolia because of the co-occurring of native and nonnative fish in Wyoming.

| Study area
We sampled 20 sites in summer 2017 in three Yellowstone River watersheds (Bighorn, Powder, and Tongue Rivers) in the Wyoming Mountain steppe, and 12 sites in two Selenge River watersheds (Delgermörön and Eg rivers) (Figure 1). Sites were chosen to maximize variability in hydrogeomorphology and ensure that sites accurately represented the broad geographic ecoregions that we sampled. Sites were selected as part of a larger macrosystem ecology project using the GIS-based tool RESonate to characterize river segments using valley-scale hydrogeomorphic variables (Williams et al., 2013). Specific stream metrics for all of our sample sites can be found in Appendix A, and geomorphologic variables for these areas can be found in more detail in Shields et al. (2021), a related project that was conducted under the same broader macrosystems project.

| Fish and benthic invertebrate collections
At each site, fishes were collected from reaches measuring 20 times the average wetted width of the stream (Patton et al., 2000).
Fishes were first collected using one-pass backpack electrofishing (ETS model: ABP-2) supplemented with hook and line and seining following electrofishing in areas where water depth or conductivity may have impacted electrofishing, following American Fisheries Society standard collection protocols (Bonar et al., 2009). To assist in areas where electrofishing success is non-optimal due to low conductivity, supplemental hook and line and seines were used. All collected fishes were identified to species, weighed (g), and measured for standard length (mm). When available, up to 10 fish for each species at each site were randomly selected and sacrificed for gut analysis. Stomachs were removed and preserved in 10% formalin for later analysis. For fishes lacking a true stomach, the anterior portion of the gut to the first bend was used as a proxy (Rybczynski et al., 2008). For all fishes, only the stomach or anterior portions of the gut was examined in an effort to minimize bias caused by digestibility of diet items (Sutela & Huusko, 2000).
A quantitative survey (abundance per m 2 ) of benthic invertebrates based on methods from Minder et al. (2020) was conducted at all study sites prior to fish collection. We collected benthic invertebrates to determine the proportional environmental abundance of each diet item for diet selectivity analyses. Benthic invertebrates were collected from three microhabitats (riffles, runs, and pools) using a Surber net, Hess sampler and a modified corer sampler (0.09, 0.03, and 0.06 m 2 , respectively, mesh size 350 μm) and five samples were collected by each microhabitat resulting in a total of 15 samplers per site . All samples were preserved in ethanol (70%) in the field and sorted and identified in the laboratory using a number of keys for aquatic macroinvertebrates (Merritt et al., 2008;Thorp & Covich, 2009). After processing, samples were grouped by sites to calculate mean invertebrate abundance per site.

| Diet analyses
Gut analysis followed procedures based on previously published works (Minder et al., 2020. Guts (esophagus to pyloric valve) were evacuated of all contents in the laboratory, and contents were examined under a dissecting scope. All items were identified to family using keys and grouped by order (Merritt et al., 2008;Thorp & Covich, 2009). Orders that represented <1% of the total number of diet items among all species were grouped into a single category, referred to as "Other." Numerical abundances of each diet item were recorded, and average proportional abundances were calculated for each order.
Calculations of frequency of occurrence (FO), and mean prey abundance (Ni) were used to quantify diets of individual fishes. FO was calculated as: where FO is the occurrence of a prey item F i divided by the number of non-empty guts (P). The metric FO describes the percentage of individuals that have consumed a specific food item. While this metric does not provide details on amounts of items consumed, it is robust to limitations of other diet analysis challenges such as differences in prey condition and presence of unidentifiable tissues (Baker et al., 2014;Buckland et al., 2017). Mean prey abundance (N i ) was used to compare feeding behavior and diet composition among fishes (Macdonald & Green, 1983).

N i was calculated as:
where N i is the mean number of prey i consumed, N ij is the number of prey i in a single predator j, and ΣN ij is the sum of all the prey in a single predator gut j.
Dietary behavior was quantified with Chesson's α selectivity index (Chesson, 1978): where r i is the proportion of the diet item consumed by an individual fish, p i is the proportional environmental abundance of the diet item at the capture site, and n is the number of prey item categories present.
If α = 1/n, the item in the diet is equal to its proportion in the environment, and we can assume that the item has been randomly selected.
If α > 1/n, then the diet item has been positively selected for, and if α < 1/n, then that diet item has been avoided. Environmental abundances for diet items were calculated for each sample site and then averaged for each fish species to ensure that site-specific selectivity was maintained.
Finally, we calculated the degree of diet overlap to assess diet similarities among fish species at a site using numerical gut content abundances. Mean proportional abundances were compared pairwise among species using Schoener's similarity index: where C is Schoener's similarity index metric, and P x,i and P y,i are the proportions of diet item i in the gut of species x and y, respectively (Schoener, 1970). This index ranges from 0 to 1 with values of 0 indicating no diet overlap and values of 1 indicating a complete overlap of diet items. Schoener's index values higher than 0.6 or lower than 0.4 are generally considered ecologically relevant (Childs et al., 1998;Muth & Snyder, 1995;Wallace Jr, 1981).

| Statistical analyses
Statistical analyses were conducted using R version 3.4.3 (R Core Team, 2017). We used non-metric multidimensional scaling (NMDS) with Bray-Curtis distance to examine relationships among fish diet contents by species. NMDS generates an ordination based on a specified number of dimensions and attempts to meet the conditions of a rank similarity matrix (Clarke, 1993). NMDS also produces stress values to quantify the effectiveness of an ordination for pattern analysis, with values below 0.2 considered to be compliant (Clarke, 1993). This method uses ranked distances and is therefore useful for data that fail to meet the assumptions of nor-  (West et al., 2003).
We also conducted an analysis of similarity (ANOSIM) to test the null hypothesis that there was no difference among the insect assemblages found in the guts of our sampled fishes. ANOSIM produces a test statistic (R) that quantifies the differences that are observed between the variables tested. R is expressed as a number between 1 and −1, which is interpreted as maximum similarity between groups and maximum dissimilarity between groups, respectively (Clarke, 1993). An R-value of 1 would indicate complete dissimilarity between two groups, an R-value of 0 is interpreted as complete similarity among groups, and a negative R-value suggests that there is more similarity between groups than within groups.

| RE SULTS
We processed a total of 471 guts from nine species across 20 sites in the Wyoming Mountain steppe and 12 sites in the Mongolia Mountain steppe (  Table 2). The three most abundant families in Mongolian fishes

| Diet contents
The frequency of occurrence (FO) of diet items in the guts of collected fishes provides the simplest quantification of diets and is resilient to errors due to diet item condition and digestions ( Figure 2). Nonnative salmonids had more diverse diets than native species. The most abundant invertebrate orders observed in diets were Ephemeroptera (56%), Diptera (53%), and Trichoptera (48%); Hydracarina (4%), and Coleoptera (16%) were the least abundant. All Mongolian fishes had lower FO scores than Wyoming fishes. Russian Weather Loach had the highest single FO value of any fish with 94% containing Diptera.
When contents were quantified by numerical abundance, we excluded debris from analyses due to its non-discrete properties ( Figure 3). In fishes collected from Mongolia, Diptera (34.8%) and Ephemeroptera (24.2%) were the two most abundant diet item orders. In Wyoming, Ephemeroptera (33.0%) and Trichoptera (21.3%) were most abundant. Gut contents were significantly different among continents, but differences were not strong (ANOSIM; R = .23, p < .01). The largest difference in assemblages was driven by the proportion of Diptera between the Wyoming (17.2%) and Mongolia (34.9%). Wyoming fishes all had relatively similar diet contents with some variation, but these differences were not significant (ANOSIM; R = .031, p = .014). Conversely, we detected differences among guts for species collected in Mongolia, these differences were significant but not strong (ANOSIM; R = .11, p = .001).

| Diet selectivity
The average environmental abundance proportions for diet items in each ecoregion results in a comparison of the invertebrate assemblages across ecoregions (Table 3). Chesson's α selectivity analysis was calculated using site-specific abundances for diet items for each individual and displayed several patterns among species (Figure 4).

| Diet overlap
Schoener's similarity index results suggested significant diet overlaps (C > 0.60) among many of the species we sampled ( Table 4).
All Wyoming fishes displayed significant within and among taxa diet overlap ( Note: Invertebrates were grouped by order, unless only a single group in that order was collected. Orders that represented <1% of the total number of diet items among all species were grouped into the "Other" category.

F I G U R E 4
Bar graph displaying the selectivity values for each of the seven diet items that were recorded for all species of fish in Wyoming and Mongolia. The diet items are Coleoptera, Diptera, Ephemeroptera, Hydracarina, Plecoptera, Trichoptera and "other." There is a line that is placed horizontally across the figure that represents organisms consuming items at amounts equal to what is found in the environment. Values above that line represent positive selection and below that line represents avoidance.
Brown Trout. Siberian Stone Loach and Common Minnow diets correlated with Coleoptera gut abundance and Russian Weather Loach correlated with Diptera gut abundance.

| DISCUSS ION
Our comparison of the fish assemblages and diets of fishes in heav-   (Copp & Vilizzi, 2004;ROBOTMAM, 1977). These microhabitat preferences likely caused the observed dietary differences and NMDS ordination positions we found for the two species.
Together, low species richness and a high proportion of nonnative species in Wyoming mountain steppe fish assemblages is indicative of invaded ecosystems (Dudgeon et al., 2006;Hermoso et al., 2011;Ross, 1991). In contrast, Mongolian mountain steppe fish assemblages had higher species richness with lower dietary overlap among species and higher levels of selectivity for diet items.
Although species richness was low in our Wyoming collections, juveniles may be acting functionally separate based on diets as in other systems (Moyle & Vondracek, 1985). However, the low sample size of juvenile fishes in our study prevented further investigation.
Our comparison of fish diets for Mongolian and the Wyoming mountain steppe regions provided information that native species in Wyoming rivers have conservation concerns from stocked nonnative species, that Mongolian rivers do not currently experience. High abundances of nonnative species and high levels of dietary overlaps in our Wyoming sites is a cause of concern for native Cutthroat Trout and overall system stability (Griffith, 1988;McHugh & Budy, 2006;Peterson et al., 2004). In contrast, fish assemblages characterizing Mongolia mountain steppe rivers were composed of only native species with distinct diets and higher selectivity values, suggesting low probability for intraspecific competition within the two groups of fish we collected (salmonids and non-salmonids). We recommend a reevaluation of any proposed plans to introduce nonnative salmonids to the area, or if introduction cannot be avoided, to require pre-and postintroduction monitoring of fish assemblages and diets to assess any impacts on native fishes (Jensen et al., 2009;Mercado-Silva et al., 2008).
We also recommend examination of fish diets in both regions during multiple seasons for greater understanding of temporal trends among fishes and to determine how piscivory varies seasonally in these areas. writing -review and editing (equal).

ACK N OWLED G M ENTS
We are grateful to the field assistance and collaboration of Robert

This project was funded by an NSF Macrosystem Biology Grant
(1442595) to M. Pyron and 10 Co-PIs.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors of this study have no competing interests to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from Dryad (https://doi.org/10.5061/dryad.rjdfn 2zhk).